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Dna sequence of genes involved in putrescine production

Putrescine (1,4- diaminobutane) is generally the most abundant biogenic amine (BA) present in wine. It is undesirable because it confers the odour of “bad breath”, “rotten meat”. Bacteria produce putrescine from ornithine via an ornithine decarboxylase (ODC), or from agmatine via the reactions of an agmatine deiminase (AgDI) and a putrescine transcarbamylase (PTC). The ODC enzyme and its odc coding gene are well characterised.

They were described in several bacteria including lactic acid bacteria (LAB) of wine. In contrast, bacterial AgDI and PTC and their corresponding genes were recently described for the first time, and they were only detected in two strains of Pseudomonas aeruginosa and Streptococcus mutans. The genes of both bacteria differ significantly in terms of sequence and arrangement.

During the sequencing of the genes of the tyramine-producing pathway of Lb. brevis IOEB 9809 we have identified the 5 extremity of an extra gene sharing similarities with genes present in the genome sequences of Pediococcus pentosaceus and Listeria monocytogenes and with the PTC gene of S. mutans, suggesting that it was a PTC gene. In P. pentosaceus and L. monocytogenes the putative PTC gene belongs to a six-gene cluster coding for enzymes potentially involved in the production of putrescine from agmatine.

To determine whether L. brevis 9809 contains the same gene cluster we have sequenced the DNA region located downstream of its putative PTC gene. We have obtained the sequence of six genes similar as those detected in P. pentosaceus and L. monocytogenes. This sequence differs from the S. mutans operon, because it contains an extra AgDI gene and the gene of a transcriptional regulator located at the 3 extremity. We have cloned, expressed, purified and characterised the two putative AgDI and a membrane transporter.

Results confirmed that one of the putative AgDI was actually an AgDI, which converts agmatine to carbamoyl-putrescine, whereas the second enzyme was inactive under our production conditions. The membrane transporter was studied by the Laboratory of Molecular Microbiology of the University of Groningen. It proved to catalyse the exchange of agmatine and putrescine.

We concluded that the six-gene cluster detected in L. brevis and also present in P. pentosaceus and L. monocytogenes is an agmatine deiminase pathway involved in putrescine production. It is related to the AgDI pathway detected in S. mutans, but it differs in terms of enzymes encoded and gene arrangements. This pathway could be widely distributed amongst LAB and contribute to the presence of putrescine in wine.

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Reported by

Faculty of Enology of Bordeaux 2 University
351, Cours de la Libération
33405 Talence
France
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